Calculation of the LOB and LOD

The limit of blank (LOB) and the limit of detection (LOD) for the ddPCR analysis were measured according to the Clinical and Laboratory Standards Institute guidelines EP17.2 and relevant reports (16–18). CfDNA templates extracted from BxPC-3 cells with wild-type KRAS were used as blank samples. Forty replicates of blank samples were detected by the ddPCR assay to calculate the LOB, which corresponds to the 95th percentile of distribution of the blank values. For LOD, cfDNA templates extracted from PANC-1 cells with mutant KRAS (c.35G>A, p.G12D) and HCT116 cells with mutant KRAS (c.38G>A, p.G13D) were diluted with the wild-type cfDNA to the expected MAFs (5, 2.5, 1.25, 0.63, 0.31, 0.16, and 0.08%), respectively. Then a series of cfDNA samples with different MAFs were detected by the ddPCR assay and repeated for at least 6 independent experiments. After consideration on the results of G12D and G13D, the LOD was defined as the MAF value in which the 95% confidence interval of all replicate detections presented values above the LOB.